The development of single gene transfer techniques for plant species is of great interest and value to plant breeders because it can be used for the rapid transfer of beneficial genetic traits to plants. Numerous methods have been developed for transferring genes into plant tissues; Agrobacterium-mediated transfer (Murai et al., 1983; Fraley et al., 1983), direct DNA uptake (Paszkowski et al., 1984; Potrykus et al., 1985), microinjection (Crossway et al., 1986), high-velocity microprojectiles (Klein et al., 1987) and electroporation (Fromm et al., 1985; Fromm et al., 1986). A general problem with most of these gene transfer techniques is that the transformed tissues, either leaf pieces or cellular protoplast, must be subjected to some regeneration steps which require a considerable amount of time before a whole plant can be obtained. This process is further complicated because tissue culture procedures have not been established for many crop species. In most cases, gene transfers into crop species have been limited to transformed callus, not whole crop plants. In addition, tissue culture procedures can result in rearrangement of the inserted DNA; or somatic mutations may occur and result in the loss or alteration of desirable genetic traits accumulated by the expertise of many years of plant breeding.
Agrobacterium-mediated gene transfers are by far the most widely used gene transfer techniques, but the use of Agrobacterium strains may be limited because they do not efficiently infect monocotyledonous cereal crop species. However, recent reports (Hooykaas-Van Slogteren et al., 1984; Hernalsteens et al., 1984; Graves and Goldman, 1986; Grimsley et al., 1987; Schafer et al., 1987; Bytebier et al., 1987) suggest that conditions exist whereby Agrobacterium strains can bind to monocotyledonous plant cells and transfer their T-DNA regions into these cells. Interestingly, the report by Graves and Goldman (1986) suggests that Agrobacteria can infect scutellar and mesocotyl cells of germinating corn (Zea mays) seeds and that the resulting plants are transformed, although these transformed plants will be sectored. This technique suggests that Agrobacterium-mediated gene transfer can be accomplished without the need of any tissue culture intermediate steps. Additional support for the transformation of mesocotyl cells of germinating seeds was obtained by Feldmann and Marks (1987) as they were able to obtain G418 resistant Arabidopsis thaliana plants by co-cultivating germinating seeds with Agrobacteria containing a binary plasmid with a plant expressible neomycin phosphotransferase (NPT) II gene in its T-DNA region.
The development of gene transfer techniques for leguminous plants is of commercial interest because it facilitates the development of new cultivars with improved disease resistance, tolerance to specific herbicides and increased nutritional value. Unfortunately, even though these dicotyledonous species are susceptible to Agrobacterium infections (Facciotti et al., 1985; Owens and Cress, 1985; Byrne et al., 1987), its use for transformation is limited due to the lack of available and efficient regeneration procedures, especially for transformed tissues.
Extension of this technique to germinating seed of leguminous plants such as Phaseolus vulgaris, the common bean, is of great importance because regeneration procedures are not available, let alone the regeneration of transformed undifferentiated tissues.
The development of simple, non-tissue culture dependent methods for transfer, stable integration, and sexual transmission of genetic material into plant species is of great interest and importance. Reports from Graves and Goldman (1986) and Feldmann and Marks (1987) present evidence that transformed whole plants can be obtained via Agrobacterium-mediated transformation of the mesocotyl cells of germinating seeds.
The process of this invention represents (1) an improvement of the Graves and Goldman (1986) technique for the transformation of the seeds of monocotyledous plants and (2) its extension to dicotyledonous plants.